Epidemiological health survey has been established as a continuous process for monitoring MIC-exposed population of Bhopal, India (Technical Report 2013; Ganguly et al. 2017). Cancer incidence in Bhopal has been reported by ICMR (National Cancer Registry Programme 2010). A questionnaire-based survey has reported the spectrum of health status (Ganguly et al. 2018b); however, the information could not be corroborated with the medical records since the records are being maintained by the health care providers. Therefore, information on risk of disease-susceptibility could not be established. Additionally, no information is present in literature on the genetic or epigenetic status of the MIC-exposed population. Moreover, scientific investigation on the long-term effects of MIC has not gained importance although it has potential to affect the future health of the victims and their progenies. In fact, assays to test the persistence of immediate effects or occurrence of new disease-onset in the long run have rarely been performed for cases of chemical exposure. MIC-exposed individuals showed severe damage in lungs and eyes as an immediate effect, which crippled the gas victims with breathlessness and blurring of vision at a relatively younger age (Ganguly et al. 2017). However, immediate genetic effects reported by individual and small studies as part of the multi-center genetic screening conducted by ICMR has not been reported. Therefore, a genetic screening was required to extract information on the present genetic status of the exposed subjects and their progeny (Ganguly and Mandal 2017; Ganguly et al. 2019).
The present screening carried out 30-years post disaster has described the incidence of clonal and non-clonal stable aberrations in circulating lymphocytes, with special emphasis on the type of alterations, development of abnormal clones and chromosomal involvement in rearrangements. The abnormalities were randomly acquired in sub-population of lymphocytes, irrespective of the DNA-content of chromosomes (Ganguly et al. 2000; Ramalho et al. 1995). Altogether, aberration per aberrant cell was significantly higher in the two exposed groups compared to unexposed population, and that was significantly higher in the severely exposed population than the moderately exposed group (Ganguly and Mandal 2017). None of the present subjects had baseline cytogenetic data collected prior to accidental exposure to MIC where a natural biological variation could significantly interfere in discerning the long-term effects of MIC.
The information on overall health of each participant appeared very general. Every participant complained about eye, respiratory and orthopedic problem. The present study population were exposed to MIC, but survived the exposure for at least an additional 30 years. Hence, one might presume that they had experienced low exposure, despite being assigned to regions of moderate or severely exposed areas. Over the past 30 years, given the lifestyles and occupations of these subjects, it is not unreasonable to assume that the observed chromosome aberrations were acquired after the MIC disaster, and in conjunction with the impact of biological and environmental confounders. Three people have died during the present study, of which one had cancer and all of them appeared with extreme hypocellular condition in their peripheral blood. Although MIC was established as a non-mutagenic agent in vitro (EHP 1987), its clastogenic effect reported in exposed population may trigger clonal development and its expansion in association with cooperating effects of mutagenic events, which may arise from biological aging of lymphocytes.
Cells with structural and numerical genetic abnormalities caused by exposure can potentially circulate through the body for several years before they are eliminated due to cell death (Hande et al. 2003). However, 30 years past the MIC-disaster, many of exposed and unexposed subjects have either passed away or at least the aberrant cells in their bloodstream can be expected to be dead. The majority of the aberrations observed in Giemsa-stained assays, for instance, such as acentric fragments, dicentrics, rings, and asymmetric chromatid exchanges are likely to be unstable and will lead to the early death of the aberrant cell. In the surviving population, it is still possible that an elevated frequency of chromosomal aberrations in peripheral lymphocytes after the disaster could have increased the current risk of cancer (Yunis 1983; Heim and Mitelman 2015). Balanced translocations, which are associated with activation of proto-oncogenes (Yunis 1983), cannot be recognized at whole genome level without G-banding or mFISH, and are usually not scored in population monitoring studies (Carrano and Natarajan 1988). The “master gene” model dictates that transcription factors (the “master genes”) are the main target for activation of translocation in acute leukemias and solid tumors (Rabbits 1994). Chromosome painting (FISH) has detected persistence of translocations in the peripheral lymphocytes of victims 15 months after a radiation accident in Brazil (Natarajan et al. 1991), in Hiroshima atomic bomb survivors several decades after exposure (Lucas et al. 1992) and also in plutonium workers (Hande et al. 2003). The frequency of translocation measured by FISH-painting 6 years later was similar to that of dicentrics measured 39 days post-exposure. However, the majority of the surveillance assays performed after chemical-exposure, though reported stable aberrations, are inconclusive on the frequency and survival of translocations, chromosomal involvement and the affected breakpoints (Ashby and Richardson 1985). The frequency of stable intra- or inter-chromosomal rearrangements studied by mFISH and/or mBAND FISH, though infrequently reported for chemical exposure, displayed power to detect such cryptic rearrangements at whole genome level (Hande et al. 2003). The present G-banding study, though enables screening of all chromosomes along with their breakpoints (which indicates possible alteration of oncogenes or tumor suppressors), may not be able to detect the cryptic rearrangements and CNVs. Therefore, the quantification of stable aberrations might have led to underestimation of aberrations, and thus, this study population could have more structural aberrations than presented in the present data. Putting together, the aberrations were mechanistically orchestrated by erroneous/inefficient DNA repair, mitotic non-disjunction due to defects in spindle assembly, DNA-replication stress, telomere shortening/dysfunction, and so on; collectively, factors of mitotic catastrophe.
In the present study, spontaneous and acquired abnormalities have largely included monosomal karyotypes, trisomy and complex rearrangements. This kind of rearrangements is frequently reported in hematopoietic stem cells of elderly individuals, myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and accelerated phase of chronic myeloid leukemia (CML) (Hasse et al. 2007; Ganguly et al. 2016; Ganguly 2017a; Ganguly and Kadam 2016; Schanz et al. 2013; Arber et al. 2016). Interestingly, some of the present abnormalities appeared as clonal. Thus, there is an obvious question whether these individuals are at risk of hematologic neoplasia. This type of aberrations was considered for bone marrow examination in individuals exposed to petroleum vapor (Hogstedt et al. 1981). One individual was observed with a reciprocal translocation between 9q34 and 22q11. It is important to mention that Ph-chromosome of t(9;22)(q34;q22) has also been reported in apparently normal individuals (Ganguly et al. 2007; Boquett et al. 2013). Hyperdiploid condition in adults is often reported to have AML (Hawkins et al. 1995). Importantly, higher frequency of trisomies of X and 8 observed in the unexposed population might have been influenced by aging, occupational, and environmental exposure and other confounders. Nonetheless, the abnormalities detected in the present study could not be correlated with ‘genetically significant dose’ (GSD) of MIC due mainly to the fact that there was no knowledge about their exposure to other xenobiotics during the past 30 years (Sobels 1982; Ganguly et al. 2017). Moreover, complete or partial deletions indicate happloinsufficiency and inactivation of tumor suppressor genes (Hande et al. 2003; Ebert et al. 2008). Altogether, the chromosomal alterations detected in the present study could not directly be correlated with MIC-exposure (Ganguly et al. 2017), which occurred 30 years ago.
In general, information available on surveillance studies in chemical exposure is scanty and inadequate, and based on inappropriate study design, insufficient sample size, and non-harmonized study protocol and interpretation of the result (Ashby and Richardson 1985). Follow-up of MIC-exposed population has not been performed since the disaster to study the elimination of aberrant cells over time. Also, the survived population did not experience severe exposure as they could overcome the acute effect and survive > 30 years after the disaster. However, the peripheral lymphocytes (study-cells) got exposed almost completely at G0 phase of the cell cycle, and peripheral blood cytogenetic assay may not detect S-phase-specific clastogens as demonstrated with formaldehyde (Fleig et al. 1982; Miretskaya et al. 1982). Rather, many of the aberrations scored are dependent on the lesions being present in the genome during its replication induced by mitogen. Delayed expression of chromosomal rearrangements and persistence of stable aberrations were reported in benzene-exposed individuals (Vainio and Sorsa 1991). Assay of sister chromatid exchanges (SCE) in the subsequent metaphases of the cells exposed in vivo at G0 has predicted its failure to remove the lesions during G0-G1 phase of the cell cycle (Wolff 1981; Ghosh 1988). It therefore seems probable that MIC-exposed cells can persist and replicate, and express aberrations once stimulated with mitogen for somatic cell division. The preliminary non-clastogenic effects of several agents such as formaldehyde, acrylonitrile, epoxy resins, etc. have been suspected of leading to carcinogenic response in humans in the long run, and continue to stimulate cancer epidemiology studies (Brewen et al. 1972).
Cytogenetic surveillance studies have discussed the life-span of radiation-induced damaged T-lymphocytes exposed in vivo in great detail. An initial rapid fall of aberrant lymphocytes was reported in several radiation-exposed individuals, followed by a slow disappearance (Ramhalo et al. 1995; Preston et al. 1974; Michie et al. 1992; McLean and Michie 1995). However, the phenomenon of loss of telomeric DNA from each cell division and replicative lifespan of damaged T-lymphocytes needs further elucidation (Röth et al. 2003). Nevertheless, daughter cells of a cell carrying breaks receive stably damaged chromosomes, and such propagation of chromosomes with stable damage would also lead to an underestimation of the death rate of T-lymphocytes. Such understanding of life-time and half-life of chemical-exposed T-lymphocytes harboring unstable and stable aberrations is limited, and no attention was ever paid during 30 years on the incidence of stable aberrations since MIC-disaster, though the accident in Bhopal was one of history’s worst disasters (Ganguly et al. 2017; Dhara et al. 2002; ICMR Technical Report 2008).
Exposure of chemical analysis is highly complex in humans, and it is practically impossible to address the interaction with lifestyle, environmental and occupational exposure to multiples of hazardous agents. The first surveillance cytogenetic screening in Bhopal population was conducted during 2015–2017. However, onset and persistence of the aberrations cannot be commented upon since the initial studies had described only breaks and chromatid exchanges in solid-stained metaphases (Ganguly et al. 2017; Ghosh et al. 1990). In fact, information on persistence of aberration in accidentally exposed population is limited in general with scanty information on chromosomal involvements at whole genome level (Lindholm et al. 1998; Pressl et al. 2000). Description of stable aberrations is generally derived from solitary/cocktail FISH or mFISH. Nevertheless, such information on persistence or acquisition of stable aberrations is meager in chemical-exposed population (Ashby and Richardson 1985). Therefore, the present report on type of structural and numerical alterations, and also on participating chromosomes along with their breakpoints might inform about the future risk in cancer epidemiology studies in MIC-exposed survivors. Although, clastogenic effect of smoking is conflicting (Crossen and Morgan 1980; Evans 1982; Ghosh et al. 1989), its carcinogenic consequences are consistent with cancer epidemiology. The present result would also be guiding factor for establishment of exposure index of chemical incidents (Ganguly 2017b), and in long-term surveillance assay. In conclusion, the present study has presented a wide spectrum of chromosomal alterations, including numerical and structural rearrangements detected from karyotypic assessment of 100 G-banded metaphases. Moreover, the classification of stable and replicable rearrangements and their presence in the present study population who were exposed to MIC 30 years ago, is noteworthy for epidemiological surveillance of their health.